In spite of prolonged, effective treatment using currently available antiretroviral therapy (ART), HIV replication continues at residual levels in patients, even below the detection limit (~50 vRNA copies per ml of plasma) of ultrasensitive clinical viral load assays. The clinical significance and the cellular source of this persistent residual viremia still remain unclear. Although residual viruses remain incapable of establishing productive infection in presence of ART, it is not clear whether they consist of genetic materials capable of producing infectious and replication-competent virus particles after therapy-interruption. If true, then the significance is that residual viruses could potentially contribute o the evolution of drug-resistance in patients during low-adherence to therapy and also spark rapid spreading of HIV when ART is discontinued. To investigate these possibilities, we have prepared this R03 application with the objectives to obtain knowledge about the replication potential, coreceptor usage and cell-tropism of residual plasma viruses present in patients on effective therapy. We will be testing two hypotheses in this application: the hypothesis 1 is that most RVs are replication-competent, and can infect and replicate readily in activated CD4 T cells in absence of ART, which we have formulated based on our preliminary data obtained from a patient on suppressive ART. The hypothesis-2 is about how residual virus can potentially establish a chronic reservoir and persist in an intrinsically stable and proliferating cell-type, perhaps in haematopoietic stem/progenitor cells of monocyte-macrophage lineage. This is formulated based on our and others published data in the field. To test our hypotheses and accomplish our overall objectives, we will pursue two specific aims: in Aim 1, we will clone residual viruses molecularly starting from residual plasma vRNAs using a novel method that we designed, and test the cloned viruses for their infectivity and coreceptor usage during infection of target cells in vitro. In Aim 2, we will determine replication potential of cloned residual virues in CD4 T cells, macrophages, CD34+ stem cells and primary astrocytes in vitro and compare with that of corresponding primary virus isolates from CD4 T cells, in order to help reveal the residual viruses' source in vivo. The rationale for the proposed research is that once we clone and characterize residual viruses phenotypically in vitro, we can put efforts to understand what role they play in drug resistance, viral load blips, persistent immune activation and sub-optimal immune recovery in patients on ART. It is our expectation that the proposed project will generate useful reagents and critical new information that will allow us to probe the clinical significance of low-level viremia during suppressive ART.